Ultrasensitive, low-input detection of avocado sunblotch viroid via RPA-CRISPR and nanopore array single-bead fluorescence readout

This study, published in Nature, presents an ultrasensitive and low input detection platform for Avocado Sunblotch Viroid (ASBVd) that integrates isothermal amplification, CRISPR-Cas12a–mediated signal conversion, and nanopore array–based single-particle analysis. The system demonstrated reliable performance across samples collected from multiple orchards and tissue types, yielding accurate positive and negative results. Compared with dLAMP assays reported in previous studies, the method exhibited higher sensitivity, detecting concentrations down to 10−3ng/μL total cDNA (1.68 copies/μL). Furthermore, each readout consumes only 40nL of the magnetic bead–fluorophore mixture from the entire CRISPR product, which is over 100-fold less than conventional fluorescence-based assays using plate readers. Unlike PCR-based approaches, the assay operates under isothermal conditions without the need for precise thermal cycling. Thereby, it reduces equipment requirements and enables rapid and portable detection, which is well-suited for field applications. With further integration and closed-cartridge development, this platform has the potential to be scaled for large-scale surveillance and adapted to detect a wide range of pathogens beyond ASBVd. 

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